The homeobox Nkx2-5, discovered in the host laboratory, encodes a key transcriptional regulator of cardiac development in vertebrates, and mutations in human NKX2-5 are associated with congenital cardiac disease. We will undertake a molecular dissection of one functional domain within Nkx2-5, a transcriptional activation domain within the C- terminus (C-TAD) which is bipartite in structure, requiring both a conserved GIRAW motif and an upstream tyrosine-rich domain. We hypothesize that C-TAD is important for Nkx2-5 activity and regulation in hear development. The proposal seeks to characterize in detail C-TAD activity in vitro and in vivo, and to isolate interacting proteins. We will: 1) perform mutagenesis of individual and multiple tyrosines to assess their importance; 2) assess whether C-TAD tyrosines are phosphorylated using tandem mass spectrometry; 3) assess whether C-TAD influences synergistic interaction act between Nkx2-5 and the cardiac transcription GAT4; 4) assess the significance of point mutations in the GIRAW motif and tyrosines for Nkx2-5 function in ES cell-derived embryoid body cultures and in chimeric mice; and 5) use the yeast 2-hybrid system to isolate interacting proteins.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32HL010389-03
Application #
6625219
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Commarato, Michael
Project Start
2001-12-01
Project End
Budget Start
2002-12-01
Budget End
2003-11-30
Support Year
3
Fiscal Year
2003
Total Cost
$40,920
Indirect Cost
Name
Victor Chang Cardiac Research Institute
Department
Type
DUNS #
752629279
City
Nsw
State
Country
Australia
Zip Code
2010
Furtado, Milena B; Solloway, Mark J; Jones, Vanessa J et al. (2008) BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4. Genes Dev 22:3037-49
Solloway, Mark J; Harvey, Richard P (2003) Molecular pathways in myocardial development: a stem cell perspective. Cardiovasc Res 58:264-77